We propose to apply the technique of Optical Detection of Magnetic Resonance (ODMR) to investigate the structural properties and the reaction mechanisms of selected enzymes. The triplet state of the flavin coenzyme will be enlisted as an intrinsic programmable spin-label, or probe, which by its very nature functions at the active site of flavoenzymes. The optical and magnetic resonance parameters of the flavin triplet state will be fully characterized. These data will then serve as a basis for investigating changes in the flavin structure and oxidation state, changes in proximate sulfhydryl groups and/or paramagnetic ions, as well as changes in protein conformation. Such studies, when combined with the identification of reaction intermediates, can provide direct evidence for the mechanism of enzyme catalyzed reactions. We propose to study the latter by using rapid-freeze techniques and other methods to trap and identify covalent adducts, initially in the amino-acid oxidase systems and subsequently in glucose oxidase and NADH dehydrogenase. By monitoring both flavin and NAD we have the capability of simultaneously interrogating both redox couples in this electron transfer system. Thus selected proteins will be investigated by ODMR in order to obtain information not accessible by other means.